The goals of this project are to identify the changes in gene expression in various testis lesions induced by known toxicants, determine which cell types and mechanisms are altered after exposure to a toxicant, and define the sequence of changes in gene expression to separate earlier from later effects in testis lesions. (1) We hypothesize that altered patterns of gene expression can be used to identify primary and secondary responses and to identify the initial target cell of a toxicant in the testis, and that the temporal pattern of changes in gene expression can be used to define the mechanisms of toxicity. We are using the NIEHS mouse microarray representing ~8700 genes expressed in a variety of tissues to determine the effects of 4 known male reproductive toxicants. They are tri-o-cresyl phosphate (TOCP), ethylene glycol monomethyl ether (EGME), Boric Acid (BA), and Cadmium (Cd). TOCP initially produces vacuoles in Sertoli cells, followed by death of early pachytene spermatocytes and round spermatids. EGME kills pachytene spermatocytes, but also produces concomitant effects on Sertoli cells. BA inhibits sperm release and is likely to act on Sertoli cells, but differently than TOCP and EGME. Cd produces divergent effects on both Leydig cells and Sertoli cells. These chemicals were chosen because they have relatively specific effects on the testis, but produce these effects at different times after initial exposure and appear to act through different routes and/or target different cell types. The present goal is to determine if changes in patterns of gene expression in the testis in response to these toxicants involves a distinct subset of genes for each toxicant, moderately overlapping subsets of genes, or a particular group of genes that responds to all 4 toxicants. Longer term goals are to determine the specific effects of different toxicants on gene expression in individual cell types of the testis, the time course of changes in gene expression induced by toxicants in different cell types of the testis, the patterns of changes in gene expression with different doses of toxicants, and the similarities and differences in the patterns of change in gene expression induced by other chemicals known or suspected to cause injury to the testis. (2) An unusually large number of unique genes, testis-specific homologs, novel members of gene families, and alternate transcripts are expressed in the testis, often during specific phases of spermatogenesis. The NIEHS mouse microarray has a low representation of the genes expressed in male germ cells and we plan to prepare a mouse testis microarray that will be considerably more informative for studies of male reproductive toxicants, as well as for studies on effects of gene knockouts, endocrine manipulations, and other treatments. Eight cDNA libraries constructed with mRNA from mouse testis and germ cells isolated at different stages of development are being subjected to single-pass sequencing from the 5' end to identify clones to be used for this purpose.